Collagen production in cardiac fibroblasts during inhibition of aminopeptidase B

Objective. To determine whether the aminopeptidase B inhibitor, arphamenine A, could affect collagen production and expression in control and TGF-B-1-treated cardiac fibroblasts. Design and Methods. Cardiac fibroblasts from passage 2 from normal male adult rats were cultured to confluency and incubated with and without 600 pmol/l TGF-beta(1) for 2 days in serum-free Dulbecco's modified Eagle's medium and then incubated with 100 mu mol/l arphamenine A for 1 day in this medium with added ascorbic acid, beta-aminopropionitrile and titriated proline. Soluble collagen was measured in the conditioned medium and non-soluble collagen in the cell layer. Aminopeptidase activity was estimated by spectrophotometric determination of the liberation of p-nitroaniline from alanine- or arginine-p-nitroanilide. Matrix metalloproteinase (MMP) and lysyl oxidase activity were assayed in the conditioned medium. A semi-quantitative reverse transcriptase-polymerase chain reaction was used to examine the expression of lysyl oxidase and collagen type I and III. Results. Arphamenine A dose-dependently inhibited basal and TGF-beta(1)-stimulated aminopeptidase activity. Arphamenine A reduced soluble and non-soluble collagen production in control and TGF-beta(1)-treated cardiac fibroblasts, while it decreased collagen type I and III expression only in TGF-B-1-treated fibroblasts. Lysyl oxidase, MMP-1 and MMP-2 activity were inhibited by arphamenine A in the conditioned media of control and TGF-B-1-treated cardiac fibroblasts. Conclusions. Our data show that the specific aminopeptidase B inhibitor, arphamenine A, reduces collagen production in cardiac fibroblasts and that this reduction is accompanied by a pronounced inhibition of lysyl oxidase.